A long range goal is to be able to detail the events that occur during the initiation of transcription of protein coding genes (transcribed by the RNA polymerase II machinery and also called class II genes) through the characterization of the proteins involved and their various protein-DNA and protein-protein interactions with the intent of formulating a general mechanism for the initiation of transcription of class II genes. To this end, the overall aim of this proposal is to continue and expand studies now in progress in my laboratory on the general protein factors that are required for the transcription of Class II genes and to elucidate some of the mechanisms that modulate transcriptional expression of these genes. The specific aims will be a detailed study of the reactions catalyzed by the general transcription factors required for transcription. These include studies on the interaction between the transcription factors, RNA polymerase II and promoter sequences through the use of techniques such as glycerol gradient centrifugation, gel filtration and protein cross linking. DNA footprinting will be used to analyze the DNA-protein interactions. Also, reagents that inhibit the transcription reaction at different stages (Sarkosyl or Heparin) will be used to define the steps at which each of the transcription factors act. I hope to be able to precisely define the order of interaction of the general transcription factors with each other and with the promoter sequences. These studies should yield basic models(s) from which the specific regulatory mechanisms of class II gene expression can be discerned at the molecular level.